Phylogeny and Biogeography of the Enigmatic Ghost Lineage

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OPEN Phylogeny and biogeography of the enigmatic ghost lineage Cylindrotomidae (Diptera, Nematocera) Iwona Kania‑Kłosok 1*, André Nel 2, Jacek Szwedo 3, Wiktoria Jordan‑Stasiło1 & Wiesław Krzemiński 4

Ghost lineages have always challenged the understanding of organism evolution. They participate in misinterpretations in phylogenetic, clade dating, biogeographic, and paleoecologic studies. They directly result from fossilization biases and organism biology. The Cylindrotomidae are a perfect example of an unexplained ghost lineage during the Mesozoic, as its sister family Tipulidae is already well diversifed during the Cretaceous, while the oldest Cylindrotomidae are Paleogene representatives of the extant genus Cylindrotoma and of the enigmatic fossil genus Cyttaromyia. Here we clarify the phylogenetic position of Cyttaromyia in the stem group of the whole family, suggesting that the crown group of the Cylindrotomidae began to diversify during the Cenozoic, unlike their sister group Tipulidae. We make a comparative analysis of all species in Cyttaromyia, together with the descriptions of the two new species, C. gelhausi sp. nov. and C. freiwaldi sp. nov., and the revision of C. obdurescens. The cylindrotomid biogeography seems to be incongruent with the phylogenetic analysis, the apparently most derived subfamily Stibadocerinae having apparently a ‘Gondwanan’ distribution, with some genera only known from Australia or Chile, while the most inclusive Cylindrotominae are Holarctic.

Cylindrotomidae Schinner, 18631 together with Limoniidae Speiser, 19092, Pediciidae Osten-Sacken, 18603, and Tipulidae Latreille, 1802 4 sensu stricto are classified within Tipuloidea Latreille, 1802 4, group of insects present in the fossil record since at least 220 Ma (Triassic)5. With only 71 extant 6 and 16 extinct species7, this smallest family within Tipuloidea is divided into two subfamilies Cylindrotominae, represented mainly in Holarctic Region, and Stibadocerinae with an example of vicariant distribution with a sister-group relationship between South American and East Asian taxa, supporting hypothesis an ‘ancestral’ trans-Pacific biota 8. According to phylogenetic synthesis based on combined morphological characters of adult, larvae and pupae, together with nuclear gene sequence data as 28S rDNA or CAD, the Cylindrotomidae are found as a sister group of Tipulidae (both being treated as subfamilies in Tipulidae in Ref.9. This group of insects is generally indicated as a sister group or being closely related to the Tipulidae by other authors 10–20. But, within Cylindrotomidae21, only the representatives of subfamily Cylindrotominae are known from fossil record. The oldest described representatives of Cylindrotominae are only known from the Paleogene (56.0–47.8 Ma) by the extant genera Cylindrotoma Macquart, 1834 22 and Diogma Edwards, 1938 23–33, plus the extinct genus Cyttaromyia Scudder, 18777,34, while the oldest stem Tipulidae are Jurassic and the oldest crown Tipulidae are Cretaceous5,35–37. Thus, the Cylindrotomidae can be considered as a typical ghost lineage during the Mesozoic. This phytophagous group of craneflies, which immatures lives among mosses and herbaceous plants. The Cylindrotomidae (Supplementary Data S1) probably knew a period of diversification during the Eocene, sufficient to become frequent enough to be found as fossils. Most of the fossil Cylindrotomidae are

1Department of Biotechnology, Institute of Biology and Biotechnology, University of Rzeszów, Zelwerowicza 4, 35‑601 Rzeszów, Poland. 2Institut Systématique Evolution Biodiversité (ISYEB), Muséum National d’Histoire Naturelle, CNRS, Sorbonne Université, Université des Antilles, EPHE, Paris, France. 3Laboratory of Evolutionary Entomology and Museum of Amber Inclusions, Department of Invertebrate Zoology and Parasitology, University of Gdańsk, 59, Wita Stwosza St, 80308 Gdańsk, Poland. 4Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Sławkowska 17, 31‑016 Kraków, Poland. *email: [email protected]

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know from the Middle Eocene Baltic amber31,32,38,39 and the Late Eocene Florissant Formation in USA24,39. Three species of Cylindrotoma are known from impressions of the Ypresian Fur Formation; two species from the Ølst Formation of Denmark were described within Cyttaromyia29 (Supplementary Table S1). Four species of Cyttaromyia were described from the Eocene Green River Formation USA, the other were described from Florissant Formation and Kishenehn Formation in USA, Middle Salt Formation in Alsace (France), Biamo Formation in Russia, and from Baltic amber24,27,28,30,31,40. Here we propose a morphological phylogenetic analysis to define the relationships between extinct genus Cyttaromyia and the other taxa in the family. We also describe two new species of Cyttaromyia on the basis of new fossils from the same Formation, and new technics of research give us possibility to redescribe Cyttaromyia obdurescens Cockerell, 192427. Results Systematic paleontology. Order Diptera Linnaeus, 175842 Infraorder Tipulomorpha Latreille, 18024 Family Cylindrotomidae Schinner, 18631 Subfamily Cylindrotominae Schinner, 1863 1 Genus Cyttaromyia Type species: C.yttaromyia fenestrate Scudder, 1877 34, by monotypy.

Key to species of the genus Cyttaromyia Scudder, 1877 34

1. Wings without distinct patterning………………..………………………….…...…...………………………3. – Distinct patterns of coloration on wings………………..………………………….…...…...…………2.

2. Rs longer than R 2+3+4 and R 3+4 combined………………..…………………...... ……….…...…...Cyttaromyia vahldieki Freiwald, 1991 29 Denmark/Ølst Formation

– Rs shorter than R2+3+4 and R3+4 combined………………..…………………………...... …...…...Cyttaromyia rayona Freiwald & Krzemiński, 1991 30 Russia/Biamo Formation

3. Wings hyaline………………..………………………….…...…...………………………………………………4.

– Wings pale brownish without conspicuous markings with end of marginal cell apically somewhat clouded (Cockerell, 1924)………………..………… ………………………… ………………………… ………………………… ………………………………….…...…...Cyttaromyia reclusa Cockerell, 1924 26 USA/Green River Formation/Roan Moutains/Colorado

4. Vein R1 well-developed………………..………………………….…...…...………………………………5.

– Vein R1 reduced………………..………………………….…...…...……………………………………7.

5. Crossvein m-cu situated beyond bifurcation of Mb on M 1+2 and M 3+4; Sc terminating in C far beyond fork of Rs………………..………………………….…...…...………………………………………………6.

– Crossvein m-cu situated at bifurcation of Mb on M1+2 and M3+4; Sc terminating in C just beyond fork of Rs………………..…………… …………………………………………...Cyttaromyia gelhausi sp. nov. USA/Green River Formation

6. Crossvein sc-r one of its length before tip of Sc; A 1 tip before tip of Sc level………………..……………………… ………………………… …………………………….…...…...Cyttaromyia obdurescens Cockerell, 192527 USA/Green River Formation/Roan Moutains/Colorado

– Crossvein sc-r at least two of its length before tip of Sc; A 1 tip beyond e tip of Sc level……………… ..………… ………………………………………….…...…...Cyttaromyia princetoniana Scudder, 189424 USA/Green River Formation

7. Sc elongate, terminating in C well beyond level of fork of Rs………………..………………………….…...…...8.

– Sc short, terminating in C before level of fork of Rs………………..…… ………………………… …………………………...... …………………….…...…...……………Cyttaromyia freiwaldi sp. nov. USA/Green River Formation

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8. Crossvein m-cu situated at most or before fork of Mb on M1+2 and M3+4… ………………………… ...... 9.

– Crossvein m-cu situated beyond fork of Mb on M 1+2 and M 3+4………………..……… ………………………… ………………………… …………………………………….…...…...Cyttaromyia frelloi Krzemiński, 1998 31 Baltic amber

9. Vein r–r (R2) terminating before level of r′–m′, before level of m–m………………..………………………….10.

– Vein r–r (R2) terminating at level of r′–m′, behind level of m–m………………..……………………… ….…... ………………………… ……………………………...Cyttaromyia fenestrata Scudder, 187734 USA/Green River Formation

10. d′-cell as long as d-cell or shorter………………..………………………….…...... …...……………11. – d′-cell longer tan d-cell………………..………………………….…...…...…………………………………12. 11. M3+4 bifurcation on M 3 and M4 approximately at level of m-m; d-cell 2× as long as M4; m-m beyond level of tip of r–r (R2)………………..………………………… ………………………… ………………………… ………………………… …………………………...... …...…...Cyttaromyia lynnae De Jong, 201940 USA/Kishenehn Formation.

– M3+4 bifurcation on M 3 and M4 before level of m–m; d-cell 1.5× as long as M 4; m–m approximately at level of tip of r–r (R2)………………..………………… ………………………… ………………………… ………………………… ………………………………….…...…...Cyttaromyia fuscula Cockerell, 192125. USA/Green River Formation

12. Fork of Mb at level of fork of Rs; d′-cell narrowed at base………………..… ………………………… …………………………...... ……………………….…...…...Cyttaromyia quievreuxi Séguy, 193428 Alsace, France/Middle Salt Formation. – Fork of Mb before level of fork of Rs; d′-cell narrow, but not narrowed at base………………..……….…..13.

13. Tip of r–r (R2) beyond level of tip of fork of M3 +4 on M3 and M4; Rs at 2× as long as R2+3+4………… ……..……………… …………………………………….…...…...Cyttaromyia scudderi Freiwald, 199129 Denmark/Ølst Formation

– Tip of r–r (R2) before level of tip of fork of M3 +4 on M3 and M4; Rs at approximately as long as 41 R2+3+4………………..……………...... …………….…...…...Cyttaromyia rossi Krzemiński, 2019 UK/Isle of Wight/Bembridge Marls

Cyttaromyia obdurescens Cockerell, 192527 (Fig. 1).

Material examined. Holotype No. 26284 (AMNH) (female); American Museum National History; Green River Formation USA, Eocene.

Emended diagnosis. Wing without color spots; Sc elongate, terminating in C well beyond level of fork of Rs, beyond r–m level but far before m–m and r′–m′ level; opposite approximately half the length of R 2+3+4; vein r–r (R2) terminating far before r′–m′ and m–m level, at level of basal part of M3; R1 well-developed; R2+3+4 longer than half length of Rs; d′-cell longer than d-cell, narrowed at its base; crossvein m-cu positioned beyond fork of Mb on M 1+2 and M 3+4; apical section of M 3 almost as long as d-cell; A 1 tip positioned near apex of wing, far behind level of Mb bifurcation on M1+2 and M 3+4, before r–m level.

Comparison. Cyttaromyia obdurescens difers from C. fenestrata, C. freiwaldi sp. nov., C. frelloi, C. fuscula, C. lynae, C. quievreuxi, C. scudderi, and C. vahldieki by a well-developed vein r–r (R2). Wing of C. obdurescens is without spot. C. rayona and C. vahldieki have diferent patterning of wings. In contrast to C. gelhausi sp. nov., crossvein m-cu is situated beyond Mb, while in C. gelhausi sp. nov., it is located at Mb bifurcation, in C. frelloi just before Mb bifurcation. In C. obdurescens, vein sc-r is located one of its length from tip of Sc, tip of A1 is located before tip of Sc, while in C. princetoniana vein sc-r is located at least two of its length before the tip of Sc, A 1 tip is located beyond level of tip of Sc. In C. obdurescens crossvein m-cu is positioned beyond fork of Mb measured from base of wing, while in C. rossi, m-cu is distinctly before fork of Mb.

Cyttaromyia gelhausi sp. nov.. http://zoobank.org/urn: lsid:zoobank.org:act:9165E3D2-514B-4524- 9F85-A9895CBF2A31. (Figs. 2, 3).

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Figure 1. Cyttaromyia obdurescens Cockerell, 192527, holotype No. 26284 (AMNH) (female): (A) habitus, lateral view; (B) apex of wing; (C) head and thorax, enlarged lateral view; (D) haltera; (E) apex of wing, drawing; (F) wing; C. terminal part of abdomen with ovipositor visible. cp, capitellum, pd, pedicellus of haltera, oc, ocellus, ped, pedicellus, pr, pronotum.

Material examined. Holotype MNHN.F.A71341 (18a, male); additional material 18b, female; 18c, female (on the same slab with holotype); Muséum national d’Histoire naturelle (MNHN), Paris; Green River Formation USA, Eocene.

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Figure 2. Cyttaromyia gelhausi sp. nov.: A.-C. No. MNHN.F.A71341 (18a, male) (holotype): (A) habitus, latero- ventral view; (B) head, ventral view; (C) hypopygium, ventral view; D. No. MNHN.F.A71341 (18c, female) (additional material), habitus, lateral view. oc, ocellus; ped, pedicellus; scp, scapus.

Etymology. Te specifc name is given to honor Doctor John Gelhaus (Academy of Natural Sciences of Drexel University), the eminent specialist on extinct and extant insects.

Diagnosis. Flagellomeres short and relatively wide; wing without color spots; Sc not very elongate, terminating in C just beyond level of fork of Rs, opposite level of crossvein r–m; opposite approximately 1/10 length of R2+3+4; vein r–r (R 2) terminating far before r′–m′ and just before m–m level, at level of basal part of M3; R1 well- developed; R2+3+4 longer than half length of Rs; d′-cell longer than d-cell, narrowed at its base; crossvein m-cu positioned at fork of Mb on M1+2 and M 3+4; apical section of M3 almost as long as d-cell.

Comparison. Cyttaromyia gelhausi sp. nov. has no distinct color patterning of the wings in contrast to C. vahldieki and C. rayona. Te wing of C. reclusa is pale brownish without conspicuous markings26, but the end of the marginal cell and the veins bounding the discal cell apically are somewhat clouded. Te body length of C. gelhausi sp. nov. is at most 6.83 mm, wing length 7 mm, while the body length of C. reclusa is 13.5 mm and wing length 12 mm. In C. gelhausi sp. nov. tibial spurs are absent while pattern of tibial spurs of C. frelloi is 1:1:231. Vein R1 is well-developed in C. gelhausi sp. nov., while in C. freiwaldi sp. nov., C. frelloi, C. fuscula, C. lynnae, C. quievreuxi, C. rossi, C. scudderi, C. vahldieki R1 is reduced. In C. gelhausi sp. nov. Sc is not very elongate, terminating in C just beyond level of fork of Rs, opposite the level of crossvein r-m, while in C. fenestrata, C. frelloi, C. fuscula, C. lynnae, C. obdurescens, C. princetoniana, C. quievreuxi, C. rayona, C. scudderi, and C. vahldieki, it is terminating far beyond level of Rs. In C. freiwaldi sp. nov. Sc terminating in C before Rs level. In C. fenestrata and C. vahldieki, Sc is very elongated, terminating in C opposite basal part of M3. Crossvein m-cu is positioned

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Figure 3. Cyttaromyia gelhausi sp. nov.: (A) No. MNHN.F.A71341 (18a, male) (holotype), wing; (B), C. No. MNHN.F.A71341 (18c, female) (additional material): (B) wing; (C) wing, drawing.

at fork of Mb on M1+2 and M 3+4 while in other fossil species of the genus Cyttaromyia this vein is situated beyond fork of Mb; in C. frelloi this vein is located before fork of Mb. Moreover, in C. vahldieki and C. fenestrata vein r–r (R2) terminating in R3+4 beyond d′ level, far beyond the level of basal part of M3, in C. gelhausi sp. nov. this vein is terminating at the level of d’, at the level of basal part of M 3.

Cyttaromyia freiwaldi sp. nov.. (Figs. 4, 5). http://zoobank.org/urn: lsid:zoobank.org:act:DB604450-AD47-4644-82E0-A33FEAEB7157.

Material examined. Holotype MNHN.F.A71342 (70A, part/68, counterpart, female), additional material 70B, female, on the same slab as holotype), Muséum national d’Histoire naturelle (MNHN), Paris; Green River For- mation USA, Eocene.

Etymology. Te new species is dedicated to the German eminent researcher Doctor Andre Freiwald (Institut für Paläontologie, Universität Erlangen).

Diagnosis. Wing without color spots; Sc short, terminating in C before level of fork of Rs, far before level of crossvein r–m; vein r–r (R2) terminating far before r′–m′ level and at m-m level, just beyond level of basal part of M3; R1 atrophied; R2+3+4 longer than half length of Rs; d′-cell shorter than d-cell, narrowed at its base; crossvein m-cu positioned beyond fork of Mb on M1+2 and M 3+4; M3 shorter than d-cell.

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Figure 4. Cyttaromyia freiwaldi sp. nov. No. MNHN.F.A71342 (70A, part, female), (holotype): (A) habitus, dorsal view; (B) head, dorsal view; (C) ovipositor, dorsal view, (D) wing venation, (E) wing venation, drawing.

Comparison. Cyttaromyia freiwaldi sp. nov. has no distinct patterning of the wings in contrast to C. vahldieki and C. rayona. In contrast to C. reclusa, the body length of C. freiwaldi sp. nov. is at most 4.8 mm, wing length 6.22 mm, while the body length of C. reclusa is 13.5 mm and wing is 12 mm long with the end of the marginal cell and the veins bounding the discal cell apically are somewhat clouded26. C. freiwaldi sp. nov. difers from other

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Figure 5. Cyttaromyia freiwaldi sp. nov.: (A) No. MNHN.F.A71342 (68, counterpart, female), (holotype), habitus, dorsal view; B.-F. No. MNHN.F.A71342 (70AB, female), (additional material): (B) habitus, dorsal view; (C) head, dorsal view; (D) terminal part of abdomen with ovipositor visible, dorsal view; (E) lef wing; (F) right wing. oc, ocelli; thx, thorax.

fossil species especially by point of termination of Sc. In C. freiwaldi sp. nov. vein Sc terminating in C before fork of Rs while in other fossil species, excluding C. reclusa27, Sc terminating in C just beyond or far beyond bifurcation of Rs. Moreover, in contrast to C. gelhausi sp. nov., C. obdurescens, C. princetoniana, and C. rayona, the vein R1 in C. freiwaldi sp. nov. is reduced. Vein r–r (R2) of C. freiwaldi is terminating in R3+4 at d-cell level while in C. fenestrata and C. rayona beyond this level, in C. vahldieki even beyond d`-cell level (Supplementary Data S2).

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Phylogenetic position of Cyttaromyia within Cylindrotomidae. Te parsimony analysis yielded three equally most parsimonious cladograms, 53 steps long, with consistency index CI = 62, RI = 66. Teir consensus majority rule cladogram is shown in Fig. 6A. Te Cylindrotomidae clade is supported by six synapomorphies: presence of petiole (character 15, state 0), relationship of R 3 and R 4 (character 17, state 1), position of crossvein m-cu relative to the bifurcation of M3+4 (character 18, state 1), shape of d-cell (character 20, state 1), position of tip of A2 (character 25, state 0), morphology of aedeagus (character 28, state 2). Cyttaromyia is supported on consensus tree by the ‘presence of supernumerary crossvein connecting vein R4+5 with M1 near its origin, to produce two discal cells’ (character 16, state 1). Te clade (Cylindrotominae + Stibadocerinae (Hennig, 43 1973) ) is supported by two synapomorphies, relationship of M 1 and M 2 (character 14, state 1), position of tip of 41 A2 (character 26, state 0). Te clade [Phalacrocera replicata Linnaeus, 1758 + (Liogma nodicornis Osten Sacken, 186544 + Triogma trisculata Shummel, 182945)] is supported by one synapomorphy, viz. position of crossvein m-cu relative to the bifurcation of Mb (character 19, state 1). Triogma trisculata appears as the sister-group to Liogma nodicornis, the clade (Liogma nodicornis + Triogma trisculata) being supported by three synapomorphies: shape of fagellomeres (character 2, state 1), position of R 5 (character 13, state 1), degree of reduction of crossvein r–m (character 24, state 1). Te clade Stibadocerinae [= (Stibadocerodes australiensis Alexander, 192246 + (Stibadocera bullans Enderlein, 191247) + (Stibadocerella pristina Brunetti, 191848 + Stibadocerina chilensis Alexander, 192849)))] is supported by two synapomorphies: the number of branches of Rs reaching wing margin (character 9, state 1), relationship of R3 and R 4 (character 17, state 2) (Supplementary Data S3). Discussion Te subfamily Cylindrotominae 21 currently contains more species and genera, compared to the Stibadocerinae. Te oldest record of the extinct genus Cyttaromyia is Paleogene, as for the genus Cylindrotoma. Te fossil record of the Cylindrotominae dated back to at least 56.0 Ma 29, with no evidences on older occurrences. Other representatives of Cylindrotominae are known in the Eocene 24,25,27,28,31,32,38, but most of them are strictly modern 6 (Supplementary Figs. S1, S2). According to our parsimony analysis (consensus tree), Cyttaromyia falls as sister group of all the extant genera of Cylindrotomidae, and thus belongs to the stem group of the family, and could correspond to a diferent subfamily. Also, the extant Cylindrotominae appear paraphyletic in respect to the Stibadocerinae because the two genera Cylindrotoma and Diogma fall in an unresolved polytomy with this subfamily plus a clade that contains the other cylindrotomine genera. Nevertheless, these results are preliminary and would need to be completed by the addition of characters, in particular molecular. Te Cylindrotominae (and also Cyttaromyia) have a Holarctic distribution, while the Stibadocerinae have a more disjunctive distribution in Indo-Malaysia, Australo-Papua and Southern Neotropics (Taiwan, China, Indonesia, Malaysia, India, Papua New Guinea, Philippines, Australia, and Chile). Such distribution resembles that of an ancient Gondwana group, with ‘relic’ taxa in Australia and Chile; but the present phylogenetic analysis would contradict this hypothesis, as the only known stem representative of the family is also Holarctic. Further analyses together with discoveries of fossil Cylindrotomidae in the Southern Hemisphere shall be necessary to clarify this complex, strange situation. From a taxonomic point of view, Architipula is characterized by the occurrence of vein Sc tip beyond fork of Rs level, subequal to or a little shorter than veins R2+3 and R3 combined, distinctly inclined crossvein m-m between 51 M1+2 and M3, usually short and straight vein A2 Cyttaromyia is characterized by the occurrence of two discal cells (d-cell and d′-cell), supernumerary crossvein r′–m′ connecting vein R 5 with M 1 near its origin, to produce two discal cells. Some similarities are present in the wing venations of Cyttaromyia and Cylindrotoma: separate M1 and M2 and relatively long vein M1. In Cylindrotoma the crossvein r′–m′ is atrophied, but the base of vein M1 is strongly arched and only one discal cell (d-cell) is present. In all other Cylindrotominae, like Diogma (recorded from the Middle Eocene)32, or other genera with a younger fossil record, the crossvein r′–m′ is reduced and only one discal cell (d-cell) is present (Fig. 6B,C; Supplementary Figs. S3, S4). Conclusion Te revision of Cyttaromyia obdurescens and the description of two new species Cyttaromyia gelhausi sp. nov. and Cyttaromyia freiwaldi sp. nov., allowed us to propose a key to the species of this genus. We have also made the frst morphological phylogenetic analysis of the Cylindrotomidae, with in the rather surprising result of the putative paraphyly of the Cylindrotominae and a position of Cyttaromyia in the stem group of this family. Material and methods Te study was based on material from the collection of the Muséum national d’Histoire naturelle (MNHN), Paris (fve specimens) and American Museum National History (AMNH) (one specimen). Te imprints from sediments of Green River Formation USA (age 50.3–46.2 Ma42) were studied using a Nikon SMZ 1500 stereomi- croscope equipped with a Nikon DS–Fi1 camera in University of Rzeszów. Te microphotographs and measure- ments were taken with NIS–Elements D 3.0 sofware. Drawings were completed by tracing the photographs, nomenclature of wing venation was used 31. Te Eocene Green River Formation USA (50.3–46.2 Ma) (Rocky Mountains, Colorado, Wyoming, Utah) is one of the most famous Eocene palaeontological sites of the World. Te sediments include mainly calcium car- bonate, calcite and aragonite. Te occurrence of diferent types of sludge varies with the geological levels. Tipton Shale Member in Greater Green River Basin is the oldest rock formation of the Green River 52. Placement of the genus Cyttaromyia within Cylindrotomidae was tested with the use of Maximum Parsi- mony (MP) criterion, implemented in TNT 1.5 sofware package, with the ‘Traditional Search’ options53,54, with memory to store 99,999 trees, 10,000 replications, with 100 trees to save per replication; utilizing

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Figure 6. (A) Consensus relationships tree of genera of subfamily Cylindrotominae. Filled circles indicate synapomorphies or autapomorphies; open circles indicate plesiomorphies. Number of character given above circles, states of characters below circles; (B) Wing venation of fossil Cylindrotominae with chronostratigraphic distribution view; (C) Wing venations of representatives of genera: Cylindrotoma, Cyttaromyia, Diogma, represented in fossil record. Wing venation redrawing 24,25,27–29,31,34,42,50. Stratigraphic chart according to International Stratigraphic Chart, International Commission of Stratigraphy (v. 2021/05) https://strat igrap hy. org/chart .

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tree-bisection-reconnection (TBR) algorithm and collapsing zero length branches. Te type species of extinct and extant genera of family Cylindrotomidae were included in the analysis. Architipula seebachi (Geinitz, 1884)55— type species of the genus Architipula Handlirsch, 1906 56, was selected as outgroup because the Architipulinae are closely related to Cylindrotominae. Tipula oleracea Linnaeus, 1758 41 was used as a type species of the genus Tipula, in the family Tipulidae, currently considered as the sister family of the Cylindrotomidae. Te morphological data to the matrix were compiled in the Nexus fle using Mesquite v. 3.61 build 92757. All 28 characters of the imagines used in the analysis were treated as unordered and unweighted (Supplementary Table S2). Equal weighting analysis (EW) was performed 53; the trees received were viewed and their features studied using Win- Clada 1.00.08 and ASADO 1.61, with Unambigous Changes Only, Fast Optimization (ACCTRA N) and Slow Optimization (DELTRAN) options57–59. Tree fles received were adjusted using Corel Draw X3 and Photo-Paint Sofware. Te 28 morphological characters of the imago observed in the fossil and recent material and used for analysis are listed below. Te data matrix given is partly based on used morphological features8,21,34,41,43–45,50,56.

Received: 28 January 2021; Accepted: 31 May 2021

References 1. Schiner, J. R. Vorlaufger Commentar zum dipterologischen Teile der Fauna austriaca. V [concl.]. Wiener Entomologische Monatschrif 7, 217226 (1863). 2. Speiser, P. 4 Orthorapha. Orthorapha Nematocera. Wissenschafliche ergebnisse der Schwedischen zoologischen expedition nach dem Kilimandjaro, dem Meru und den umgebenden Massaisteppen Deutsch-Ostafrikas 1905–1906, unter leitung von prof. dr. Yngve Sjostedt. Diptera 10, 31–65 (1909). 3. Osten Sacken, C. R. New genera and species of North American Tipulidae with short palpi, with an attempt at a new classifcation of the tribe. Proc. Acad. Nat. Sci. Phila. 1859, 197–254 (1860). 4. Latreille, P. A. Histoire naturelle, generale et particuliere, des Crustaces et des Insectes. Tome troisieme. Ouvrage faisant suite a lhistoire naturelle generale et particuliere, composee par Leclerc de Bufon, et redigee par C.S. Sonnini, membre de plusieurs societes savantes. Familles naturelles des genres, Paris, 13–467. I–xii (1802). 5. Krzemiński, W. Tipula (s. lato) eva n. sp. from Cretaceous (East Asia)—Te oldest representative of the family Tipulidae (Diptera, Polyneura). Acta Zool. Crac. 35, 43–44 (1992). 6. Oosterbroek, P. Catalogue of the Crane–fies of the World. (Diptera: Tipuloidea: Pedicidae, Limoniidae, Cylindrotomidae, Tipuli- dae). https://ccw.naturalis.nl/ Accessed 28 May 2020 (2020). 7. Evenhuis, N. L. Catalog of the fossil fies of the world (Insecta: Diptera) website. Version. 2.0. http://hbs. bisho pmuse um. org/ fossi lcat/ Accessed 13 Feb 2014 (2014). 8. Ribeiro, G. C. Te Neotropical genus Stibadocerina Alexander and its phylogenetic relationship to other Stibadocerinae genera: Further evidence of an ancestral trans-Pacifc biota (Diptera: Cylindrotomidae). Syst. Entomol. 34, 324–333 (2009). 9. Petersen, M. J., Bertone, M. A., Wiegann, B. M. & Courtney, G. W. Phylogenetic synthesis or morphological and molecular data reveals new insights into the higer level classifcation of Tipuloidea (Diptera). Syst. Entomol. 35, 526–545 (2010). 10. Alexander, C. P. Notes on the genus Dicranoptycha Osten Sacken (Tipulidae, Diptera). Entomol. News 30, 19–22 (1919). 11. Alexander, C. P. New or little-known Tipulidae (Diptera). III. Ethiopian species. Ann. Mag. Nat. Hist. 9(5), 465–472 (1920). 12. Savchenko, E. N. Tipulidae. Fauna Ukrainy 14, 1–551 (1966). 13. Savchenko, E. N. Limoniidae of South Primorye. Akademii Nauk Ukrainskoy SSR, Kiev 1–156 (1983). 14. Brodo, F. A revision of the genus Prionocera and the phylogeny of the family Tipulidae (Diptera). PhD Dissertation, Carleton University, Ottawa, Ontario (1984). 15. Oosterbroek, P. & Teowald, B. Phylogeny of the Tipuloidea based on characters of larvae and pupae (Diptera, Nematocera), with an index to the literature except Tipulidae. Tijdschrif voor Entomologie 134, 211–267 (1991). 16. Starý, J. Phylogeny and classifcation of Tipulomorpha, with special emphasis on the famiy Limoniidae. Acta Zool. Crac. 35, 11–36 (1992). 17. Ribeiro, G. C. Te phylogeny of the Limnophilinae (Limoniidae) and the early evolution of the Tipulomorpha (Diptera). Invertebr. Syst. 22, 627–694 (2008). 18. Zhang, X. et al. Comparative Mt genomics of the Tipuloidea (Diptera: Nematocera: Tipulomorpha) and its implications for the phylogeny of the Tipulomorpha. PLoS ONE 11(6), 1–20 (2016). 19. Lukashevich, E. D. & Ribeiro, G. C. Mesozoic fossils and the phylogeny of Tipulomorpha (Insecta: Diptera). J. Syst. Paleontol. 17, 635–652 (2019). 20. Kang, Z., Zhang, X. & Yang, D. Characterization of the complete mitochondrial genome of the snow crane-fy Chionea crassipes gracilistyla (Diptera, Tipuloidea, Limoniidae) with phylogenetic analysis. Mitochondrial DNA (B) 4, 2662–2663 (2019). 21. Osten Sacken, C. R. Monographs of the Diptera of North America. Part IV. Smithson. Misc. Collect. 8(219), 345 (1869). 22. Macquart, P. J. M. Histoire naturelle des insectes. Diptères. Tome première. N.E. Roret Paris. 578 (1834). 23. Edwards, F. W. On the British Lestremiinae, with notes on exotic species—3. (Diptera, Cecidomyiidae). Proc. R. Entomol. Soc. Lond. (B) 7, 102–108 (2009). 24. Scudder, S. H. Tertiary Tipulidae, with special reference to those of Florissant, Colorado. Proc. Am. Philos. Soc. 32, 163–245 (1894). 25. Cockerell, T. D. A. Eocene insects from the Rocky Mountains. Proc. U.S. Natl. Mus. 57, 233–260 (1921). 26. Cockerell, T. D. A. Fossil insects in the United States National Museum. Proc. U. S. Natl. Mus. 64(13), 1–15 (1924). 27. Cockerell, T. D. A. Plant and insect fossils from the Green River Eocene of Colorado. Proc. U. S. Natl. Mus. 66(19), 1–13 (1925). 28. Séguy, E. Un nouveau Cylindrotomine fossile (Tipulidae). Encycl. Entomol. (B) II(7), 47–48 (1934). 29. Freiwald, A. Insekten aus der Fur-Formation von Dänemark (Moler, oberes Paleozän/ unteres Eozän). 5. Cylindrotomidae (Diptera: Tipulomorpha). Meyniana 43, 97–123 (1991). 30. Freiwald, A. & Krzemiński, W. Cylindrotomidae (Diptera, Tipulomorpha) from the Paleogene of Bolshaya Svetlovodnaya (eastern Asiatic USSR). Paläontol. Z. 65, 339–344 (1991). 31. Krzemiński, W. Cyttaromyia frelloi sp. n., the frst representative of the family Cylindrotomidae in Baltic amber (Diptera: Tipulo- morpha). Polskie Pismo Entomologiczne 67, 303–308 (1998). 32. Podenas, S. A new species of Diogma Edwards, 1938 (Diptera, Cylindrotomidae) from Baltic amber (Eocene). Trans. Am. Entomol. Soc. 126(1), 103–107 (2000). 33. Brodo, F. A review of the subfamily Cylindrotominae in North America (Diptera: Tipulidae). Univ. Kansas Sci. Bull. 47, 71–115 (1967). 34. Scudder, S. H. Te frst discovered traces of fossil insects in the American tertiaries. Bull. U. S. Geol. Geogr. Surv. Territ. 3, 741–762 (1877).

Scientifc Reports | (2021) 11:13916 | https://doi.org/10.1038/s41598-021-91719-w 11 Vol.:(0123456789) www.nature.com/scientificreports/

35. Krzemiński, W. & Ansorge, J. New Upper Jurassic Diptera (Limoniidae, Eoptychopteridae) from the Solnhofen lithographic limestone (Bavaria, Germany). Stuttgarter Beiträge zur Naturkunde (B) 221, 1–7 (1995). 36. Lukashevich, E. D. Limoniidae (Diptera) in the Upper Jurassic of Shar Teg, Mongolia. Zoosymposia 3, 131–154 (2009). 37. Krzemiński, W., Kopeć, K. & Kania, I. New and little known species from the genus Leptotarsus Guerin-Meneville, 1831 (Diptera: Tipulidae) from the Lower Cretaceous of Northern Brazil. Cretac. Res. 78, 103–108 (2017). 38. Loew, H. Ueber den Bernstein und die Bernsteinfauna. Programm der K. Realschule Meseritz 1–44 (1850). 39. Krzemiński, W. Revision of the fossil Cylindrotomidae (Diptera, Nematocera) from Florissant and White River, USA. Paläontol. Z. 65, 333–338 (1991). 40. Krzemiński, W. et al. True fies (Insecta: Diptera) from the late Eocene insect limestone (Bembridge Marls) of the Isle of Wight, England, UK. Earth Environ. Sci. R. Soc. Edinb. 110, 495–554 (2019). 41. Linnaeus, C. Systema Naturae per Regna tria Naturae, secundum classes, ordines, genera, species, cum characteribus, diferentiis, synonymis, locis. T. 1. Holmiae: Impensis Direct. Laurentii Salvii. 800 (1758). 42. Greenwalt, D. E. et al. Diptera of the middle Eocene Kishenehn Formation. I. Documentation of diversity at the family level. Palaeontol. Electron. 22, 1–56 (2019). 43. Hennig, W. Flügelgeäder und System der Dipteren unter Berücksichtigung der aus dem Mesozoikum beschriebenen Fossilien. Beiträge zur Entomologie 4, 245–388 (1973). 44. Osten Sacken, C. R. Description of some new genera and species of North American Limnobina. Part I. Proc. Entomol. Soc. Phila. 4, 224–241 (1865). 45. Schummel, T. E. Beschreibung der in Schlesien einheimischen Arten einiger Dipteren-Gattungen 1 Limnobia. Meigen. Beitrage zur Entomologie, besonders in Bezug auf Schlesien, Breslau 1, 97–201 (1829). 46. Alexander, C. P. New or little-known Tipulidae (Diptera) IX. Australasian species. Ann. Mag. Nat. Hist. 9(9), 297–315 (1922). 47. Enderlein, G. 1912. Die Phoridenfauna Südbrasiliens. Stettiner Entomologische Zeitung 73, 16–45 (1938). 48. Brunetti, E. Revision of the Oriental Tipulidae with descriptions of new species. Part II. Rec. Indian Mus. 15, 255–344 (1918). 49. Alexander, C. P. Diptera. Fam. Tipulidae. Subfam. Cylindrotominae. Genera Insectorum. 187(1927), 1–16 (1928). 50. Meigen, J. W. Systematische Beschreibung der bekannten europäischen zweifügeligen Insecten. I. Friedrich Wilhelm Farstmann, Aachen, i–xxxvi+1–332+[1] (1818). 51. Kopeć, K., Krzemiński, W., Skowron, K. & Coram, R. Te genera Architipula Handlirsch, 1906 and Grimmenia Krzemiński & Zessin, 1990 (Diptera: Limoniidae) from the Lower Jurassic of England. Palaeontol. Electron. 20.1.15A, 1–7 (2017). 52. Self, J. G., Johnson, R. C., Brownfeld, M. E. & Mercier, T. J. Stratigraphic cross sections of the Eocene Green River Formation in the Piceance Basin, northwestern Colorado: US. Geol. Surv. Digit. Data Ser. DDS-69-Y 5, 7 (2010). 53. Golobof, P. A., Farris, J. & Nixon, K. TNT, a free program for phylogenetic analysis. Cladistics 24, 774–786 (2008). 54. Golobof, P. A. & Catalano, S. TNT version 1.5, including a full implementation of phylogenetic morphometrics. Cladistics 32, 221–238 (2016). 55. Geinitz, F. E. Über die Fauna des Dobbertiner Lias. Zeitschrif der Deutschen Geologischen Gesellschaf 36, 566–583 (1884). 56. Handlirsch, A. Die fossilien Insecten und die Phylogenie der rezent Formen. Engelmann, Leipzig, (1906–1908). 57. Maddison, W. P. & Maddison, D. R. Mesquite: a modular system for evolutionary analysis. Version 3.61. http://mescuiteproject. org. Accessed 22 June 2020 (2009). 58. Nixon, K. C. WinClada ver. 1.00.08 Published by the author, Ithaca, New York (2002). 59. Nixon, K. C. ASADO, version 1.85 TNT-MrBayes Slaver (vl 5.30). Published by the author, Ithaca, New York (2004). Acknowledgements We would like to acknowledge for fnancial support of these research by JM Rector of University of Rzeszów (Al. Rejtana 16c, 35-601 Rzeszów). We would like to thanks two anonymous Reviewers for valuable comments and remarks. Author contributions I.K.-K. conceived and designed the study, lead and performed the data analysis, interpretations and writing, making photographs, graphical fgures, analysis; correspondence. A.N. writing and corrections of the manuscript. J.S. corrections of phylogenetic analysis methods. W.J.-S. making photographs and drawing, graphical fgures, analysis. W.K. corrections of the manuscript. Funding Open Access funding enabled and organized by the University of Rzeszów.

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Scientifc Reports | (2021) 11:13916 | https://doi.org/10.1038/s41598-021-91719-w 13 Vol.:(0123456789) PHYLOGENY AND BIOGEOGRAPHY OF THE ENIGMATIC GHOST LINEAGE CYLINDROTOMIDAE (DIPTERA, NEMATOCERA)

Iwona Kania-Kłosok1*, André Nel2, Jacek Szwedo3, Wiktoria Jordan-Stasiło4, Wiesław Krzemiński5

Supplementary information Supplementary Data S1 Cylindrotomidae is represented by nine extant genera: Cylindrotoma, Diogma, Liogma, Phalacrocera, Stibadocera, Stibadocerella, Stibadocerina, Stibadocerodes[49], and Triogma. The Stibadocerinae comprise the four genera Stibadocerodes, Stibadocera, Stibadocerella, and Stibadocerina. The Cylindrotominae contain the genera Cylindrotoma, Cyttaromyia, Diogma, Liogma, Phalacrocera, and Triogma.

Supplementary Data S2 Cyttaromyia obdurescens Cockerell, 1925[27] (Fig. 1)

Redescription. Head (Fig. 1A, C) small, not well-preserved, eyes well-developed, large, occiput dark brown colored. Thorax brown (Fig. 1A) only approximately 1.5x as long as head, pronotum dark-brown. Sc (Fig. 1B, E, F) terminating in C well beyond level of fork of

Rs, opposite approximately half length of R2+3+4; crossvein sc-r approximately one of its length from tip of Sc; R2+3+4 only shorter than d-cell and d`-cell; R3+4 longer than d-cell and d`-cell; section of R5 from r'-m' connection to margin of wing shorter than d`-cell about 0.2x of length of this section; R5 evenly curved towards margin wing; crossvein r-m positioned d- cell slightly distad of fork of Rs; four medial branches of Mb present; d-cell elongate, gradually widening towards wing margin, d-cell longer than apical sections of M1 – M4; d-cell set noticeably more basad than supernumerary cell (d'-cell), approximately 3.5x as long as wide; d'-cell noticeably narrows basad, approximately 5.5x as long as wide; crossvein m-cu located beyond fork of Mb on M1+2 and M3+4; base of apical part of M1 almost perpendicular to M2, before turning towards apex of wing; M1 (excluding base of M1, before turning towards apex of wing) and M2 almost equal in length, slightly shorter than M3 and slightly longer than

M4; apical section of M3 subsinuous from d-cell towards wing margin; distance between tip of

R5 and tip of M1 or tip of M1 and tip of M2 almost equal in length; distance between tip of M3 and M4 2x as long as distance between tip of M2 and M3; distances between tip of M2 and M4 and between tips of M4 and Cu almost equal in lengths. A2 elongate, slightly sinusoidal. Tip of

A1 positioned behind fork of Rs. Haltera (Fig. 1D) elongate, shorter than 0.25x of fore wing.

Abdomen (Fig. 1G) approximately 5x as long as thorax, ovipositor not very elongate.

Cyttaromyia gelhausi sp. nov. http://zoobank.org/urn: lsid:zoobank.org:act:9165E3D2-514B-4524-9F85-A9895CBF2A31 (Figs 2 – 3) Description. Body length 6.24 – 6.83 mm (male) (Fig. 2A), 6.72 mm (female) (Fig. 2D), wing length 7 mm (male) (Fig. 2A, 3A-C), 6.89 mm (female) (Fig. 2D). Head 1.10 – 1.93 mm wide (male) (Fig. 2A, B), 1.5 mm (female), small, not well-preserved, compound eyes well- developed, large; occiput dark brown colored; last segment of antennae rather elongate, slightly longer than penultimate one; antenna poorly preserved; palpi not visible. Thorax brown (Fig. 2A) approximately 2.5x as long as head, pronotum dark-brown. Wing length 6.59 mm, width 1.48 mm (male), 6.35 – 7.47 mm, width 1.50 – 1.70 mm (female) with a pronounced stigma in distal part of wing; Sc (Fig. 3A-C) terminating in C just beyond level of fork of Rs, opposite approximately 1/10 length of R2+3+4; position of crossvein sc-r approximately one of its length from tip of Sc; R2+3+4 shorter than d-cell and d`-cell; R3+4 longer than d-cell and d`-cell; section of R5 from r'-m' connection to margin of wing shorter than d`-cell about 0.25x length of this section; R5 evenly curved towards margin wing; crossvein r-m situated slightly distad of fork of Rs; four medial branches of Mb presented; d- cell elongate, gradually widening towards wing margin, d-cell longer than apical sections of

M1 – M4; d-cell set noticeably more basad than supernumerary cell (d'-cell), approximately 2.5x as long as wide; d'-cell noticeably narrows basad, approximately 3.5x as long as wide; crossvein m-cu located at fork of Mb on M1+2 and M3+4; base of apical part of M1 almost perpendicular to M2, before turning towards apex of wing; M1 (excluding base of M1, before turning towards apex of wing) and M2 almost equal in length, slightly shorter than M3 and slightly longer than M4; apical section of M3 subsinuous from d-cell towards wing margin; distance between tip of R5 and tip of M1 or tip of M1 and tip of M2 almost equal in length; distance between tip of M3 and M4 2x as long as distance between tip of M2 and M3; distance between tip of M2 and M4 longer than distance between tips of M4 and Cu. Haltera elongate, shorter than 0.25x of wing.

Abdomen (Fig. 2A, C, D) approximately 2.5x as long as thorax, ovipositor not very elongate, 1.37 mm long, about 0.3x length of abdomen; hypopygium 1.09 mm long, wide, rather short, gonocoxites wide, massive.

Cyttaromyia freiwaldi sp. nov. (Figs 4 – 5) http://zoobank.org/urn: lsid:zoobank.org:act:DB604450-AD47-4644-82E0-A33FEAEB7157 Description. Body (Figs 4B; 5A, B) length 4.8 mm, wing length 6.14 – 6.22 mm (Figs 4B, C, D; 5 A, B, E, F). Head (Figs 4A, B; 5C) 1.33 mm wide, compound eyes well-developed, large, occiput dark brown colored; antenna rather short, not very well preserved, 2.32 – 2.36 mm; palpi not well visible. Thorax brown (Figs 4B; 5A, B) approximately 2.5x as long as head, pronotum brown. Wing length 6.14 – 6.22 mm, with a pronounced stigma in distal part of wing; Sc (Fig. 4C, D) terminating in C just beyond level of fork of Rs, opposite approximately 1/10 the length of

R2+3+4; crossvein sc-r approximately one of its length from tip of Sc; R2+3+4 shorter than d-cell and d`-cell; R3+4 longer than d-cell and d`-cell; section of R5 from r'-m' connection to margin of wing shorter than d`-cell about 0.2x length of this section; R5 evenly curved towards margin wing; crossvein r-m positioned slightly distad of fork of Rs; four medial branches of Mb presented; d-cell elongate, gradually widening towards wing margin, d-cell longer than apical sections of M1 – M4; d-cell set noticeably more basad than supernumerary cel (d'-cell), approximately 3.5x as long as wide; d'-cell only slightly narrows basad, approximately 3.5x as long as wide; crossvein m-cu located beyond fork of Mb on M1+2 and M3+4 in 0.2 length of

M3+4; base of apical part of M1 almost perpendicular to M2, before turning towards apex of wing; M1 (excluding base of M1, before turning towards apex of wing) and M2 almost equal in length, longer than M3 and M4; apical section of M3 almost straight from d-cell towards wing margin; distances between tip of R5 and tip of M1 or tip of M1 and tip of M2 almost equal in length; distance between tip of M2 and M3 1.5x as long as distance between tip of M3 and M4; distance between tip of M2 and M4 longer than distance between tips of M4 and Cu. Haltera elongate, shorter than 0.2x of wing.

Abdomen (Figs 4B; 5A, B, D) approximately 5x as long as thorax, ovipositor not very long, 2.4 mm long, about 0.25x length of abdomen.

Supplementary Data S3 Characters scored for phylogenetic analysis. 1. Proportion between length of antenna and length of body: antennae one-third body length or shorter (0); antennae comparatively long, longer than one-third body length (1). 2. Shape of flagellomeres: not serrate (0); serrate (1). 3. Proportion between length and width of first and second flagellomeres: less than 3x longer than wide (0); more than 3x longer than wide (1).* 4. Coloration of wing: hyaline (0); at least partially pigmented brownish (1). 5. Length of vein Sc: Sc rather elongate, terminate opposite fork of Rs level or beyond (0); Sc short, terminate before fork of Rs level (1). 6. Tip of Sc: Sc reaching wing margin (0); Sc partially atrophied, not reaching wing margin (1); tip of Sc completely atrophied, not reaching wing margin (2). 7. Crossvein sc-r: well-developed (0); tendency to atrophy (1). 8. Branches of radial vein Rb: four branches merging into wing apex (0); three branches merging into wing apex (1); two branches merging into wing apex (2). 9. Number of branches of Rs reaching wing margin: at least two (0); one (1).

10. Length of vein R1: R1 terminating beyond level of fork of M4 and M3 (0); R1 terminating at level of fork of M4 and M3 (1).

11. Vein R5: well-developed (0); atrophied (1).

12. Shape of base of R2+3+4: slightly waved (0); strongly curved (1).

13. Position of R5: R5 separated from point of connection of r-m and M1+2 (0); R5 separated from margin of d-cell (1).

14. Relationship of M1 and M2: at least partially separate (0); fused (1). 15. Presence of petiole: absent (0); present (1).

16. A supernumerary crossvein r`-m` connected vein R5 with M1 near its origin, to produce two discal cells: absent (0); present (1).

17. R3 and R4: separate (0); fused (1).

18. Position of crossvein m-cu relative to bifurcation of M3+4: m-cu located at fork of M3+4 (0); m-cu located at extreme base of discal cell or before it (1). 19. Position of crossvein m-cu relative to bifurcation of Mb: beyond fork of Mb (0); at fork of Mb (1). 20. Shape of d-cell: rhomboid (0); rectangular (1).

21. Position of medial veins: M1+2 or M2 and M3 separated (0); M1+2 and M3 having at least a point of contact at base (1).

22. Shape of base of M3: not indented (0); strongly indented (1).

23. The distance between tip of M4 and tip of Cu: narrow (0); very wide (1). 24. Crossvein r-m: well-developed (0); reduced (1).

25. Position of A2 tip: beyond Rs origin level (0); before of Rs origin level (1). 26. Position of A1 tip: at or beyond of d-cell level (0); before of d-cell level (1). 27. Gonostylus: two-branched with well-developed clasper and lobe (0); two-branched, but with reduced lobe (1); with a single branch (2).* 28. Aedeagus: single (0); bifid (1); trifid (2).*

Supplementary tables Supplementary Table S1. List of fossil species of Cyttaromyia (partially after used information from [26, 40]).

SPECIES AGE (MA)* TYPE HORIZON

Cyttaromyia fenestrata 37.2 – 33.9 USA/Green River Formation

Cyttaromyia frelloi 38.0 – 33.9 Baltic amber

Cyttaromyia fuscula 37.2 – 33.9 USA/Green River Formation

Cyttaromtia lynnae 46.6 – 45.8 USA/Kishenehn Formation USA/Green River Formation/Roan Cyttaromyia obdurescens 50.3 – 46.2 Moutains/Colorado Cyttaromyia princetoniana 37.2 – 33.9 USA/Florissant Formation

Cyttaromyia quievreuxi 33.9 – 28.1 France/Middle Salt Formation

Cyttaromyia rayona 38.0 – 33.9 Russia/Biamo Formation USA/Green River Formation/Roan Cyttaromyia reclusa 50.3 – 46.2 Moutains/Colorado Cyttaromyia rossi 38.0 – 33.9 UK/Isle of Wight/Bembridge Marls

Cyttaromyia scudderi 56.0 – 47.8 Denmark/Ølst Formation

Cyttaromyia vahldieki 56.0 – 47.8 Denmark/Ølst Formation * Ages are given after [6, 41]

15 16 17 18 19 20 21 22 23 24 25 26 27 28 Architipula seebachi 1 0 0 0 0 0 0 0 0 0 1 1 ? ? Cylindrotoma distinctissima distinctissima 0 0 1 1 0 1 0 0 0 0 1 0 2 2 Cyttaromyia fenestrata 0 1 1 1 0 1 0 0 0 0 0 1 ? ? Diogma glabrata 0 0 1 1 0 1 0 0 0 0 0 0 0 1 Liogma nodicornis 0 0 1 1 1 1 0 0 0 1 0 1 2 2 Phalacrocera replicata 0 0 1 1 1 1 1 0 0 0 0 0 2 2 Triogma trisulcata 0 0 1 1 1 1 0 0 0 1 0 0 2 2 Tipula oleracea 1 0 0 0 0 0 0 0 0 0 1 1 0 0 Stibadocera bullans 0 0 2 1 0 1 0 1 1 0 0 0 1 2 Stibadocerella pristina 0 0 2 1 0 1 0 1 1 0 0 1 0 2 Stibadocerina chilensis 0 0 2 1 0 1 0 1 1 0 0 1 2 2 Stibadocerodes australiensis 0 0 1 1 0 1 0 1 1 0 0 0 0 2

1 2 3 4 5 6 7 8 9 10 11 12 13 14 Architipula seebachi ? ? ? 0 0 0 0 0 0 0 0 0 0 0 Cylindrotoma distinctissima distinctissima 0 0 0 0 0 1 1 2 0 0 0 0 0 0 Cyttaromyia fenestrata ? 0 ? 0 0 0 0 2 0 0 0 0 0 0 Diogma glabrata 0 0 0 0 0 2 0 2 0 0 0 1 0 1 Liogma nodicornis 0 1 0 0 1 0 0 2 0 1 0 1 1 1 Phalacrocera replicata 0 0 0 1 1 2 0 1 0 0 0 0 0 1 Triogma trisulcata 0 1 0 0 0 1 1 1 0 1 0 1 1 1 Tipula oleracea 0 0 1 1 1 2 0 0 0 0 0 0 0 0 Stibadocera bullans 1 0 1 0 1 2 0 1 1 1 1 0 0 1 Stibadocerella pristina 1 0 1 0 1 0 0 2 1 0 1 0 0 1 Stibadocerina chilensis 1 0 1 0 1 0 0 2 1 0 1 0 0 1 Stibadocerodes australiensis 1 0 1 0 1 0 0 2 0 0 1 0 0 1 Supplementary Table S2. Matrix of characters

Supplementary figures

Supplementary Figure S1. Wing venation and color pattern of fossil Cylindrotominae with chronostratigraphic distribution view, wing venation redrawn[24, 25, 27-29, 31, 34, 40, 48]. Stratigraphic chart according to International Stratigraphic Chart, International Commission of Stratigraphy (v. 2021/05) https://stratigraphy.org/chart.

Supplementary Figure S2. Chronostratigraphic distribution of Cylindrotomidae. In white figures the number of species is given. Lines mark time of duration of the genus. Stratigraphic chart according to International Stratigraphic Chart, International Commission of Stratigraphy (v. 2021/05) https://stratigraphy.org/chart.

Supplementary Figure S3. Wing venation of species of Cyttaromyia, redrawn[28-31, 39, 40]. Abbreviations: lines cut the points: grey line – tips of A1 or A2; red line – tip of Sc; yellow line – to tip of r-r (R2).

Supplementary Figure S4. Wing venation of type species of Cylindrotominae and the genus Architipula – as an

[39,49, 60] outgroup used in parsimony analysis, redrawn . Abbreviations: lines cut the points: grey line – tips of A1 or

A2; red line – tip of Sc; yellow line – to tip of r-r (R2).

Supplementary references 60. Alexander, C.P. Diptera of Patagonia and South Chile. Part I. Crane-flies (Tipulidae, Trichoceridae, Tanyderidae). Diptera of Patagonia and South Chile, 1, 1-240 (1929).